SUPEROXIDE ANION IS IMPLICATED IN THE REGULATION OF PP2AB56δ-MEDIATED DEPHOSPHORYLATION OF BCL-2 IVAN LOW CHERH CHIET [BSc. (Hons.), NUS] A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Declaration I hereby declare that this thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. ________________________ Ivan Low Cherh Chiet 07 Nov 2012 ACKNOWLEDGEMENTS Four years of fulfilling and enriching experience; of joy, excitement and laughter; of sorrows, disappointments and despair. It was unique, but none possible without the company and contributions of these people. My utmost gratitude goes to them. To Prof Shazib Pervaiz – a supervisor, a boss and also a friend. It is your relentless guidance, enthusiasm and support that have brought me thus far. The scientific and intellectual brain teasers imparted throughout the course of my PhD is amongst the biggest drivers underlying what I have achieved today. I sincerely thank you, not only for the intellectual contributions, but also the countless opportunities that you have created for me. And not to forget, the wonderful experience we have shared over numerous conference trips as well as the time spent running together along the roads, bahut shukriya. To my thesis advisory committee (TAC) members, Prof David Marc Virshup and Assoc. Prof Marie-Veronique Clement. Thank you for the ever critical and constructive inputs, as well as the reagents and experimental tools that you have generously shared with me. Merci. To my lab mates, Tini and Maj, for tolerating my last minute orders and my irritating pesters over urgent orders, terima kasih. To Jay, for being the lab’s mother since I have first stepped into the lab as an innocent honours student, dhanyavad. To Stephen, for taking all my scoldings and naggings positively, even though they often fall on deaf ears, toh cheh. To Pat, Serena, Angeline, Carolyn, Zhou Ting and the rest of my ex- and current lab mates, thanks for sharing the wonderful working hours as well as the limited lab space and equipment with me. Thank you all! i To my hall and running friends, especially Kuan Thye, Teik Zhen, Aaron, Roonz, Eugene Tan, twin bro Lee, Huat and Alan. Thank you for spicing up my life other than lab, research, studies and work. To my parents and my younger sister, thank you for the guidance, patience, concern and support since my birth, and throughout the entire period when I am in Singapore. Mum and dad, the baby that had once cried off your sleeping hours as well as the boy that had frequently gotten your blood pressure raised hopes that he could you proud here. Last but not least, my wife, Hui Ting, words cannot describe the gratitude I have for all the support, love and care that you have rendered unconditionally. It is you that kept me going when I am down, defeated, and injured, but I am never out all because of you. Thanks for sharing my joy and happiness, my sorrows and hardships. Through thick and thin, I love you. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS . i TABLE OF CONTENTS iii SUMMARY . ix LIST OF TABLES xi LIST OF FIGURES xi LIST OF ABBREVIATIONS . xv INTRODUCTION . 1 Reactive Oxygen Species – An overview 1.1 Superoxide anion (O2-) 1.2 Hydrogen Peroxide (H2O2) . 1.3 Nitric Oxide (NO) . 1.4 Peroxynitrite (ONOO-) 10 Cellular Antioxidant Defences . 12 2.1 Superoxide Dismutases . 12 2.2 Catalase . 13 2.3 Gluthathione Peroxidases 14 2.4 Peroxiredoxins and Thioredoxins . 15 Apoptotic Cell Death 16 3.1 Caspases – the mastermind of apoptosis . 17 3.2 Extrinsic pathway 18 3.3 Intrinsic pathway . 19 3.4 The anti-apoptotic B Cell Lymphoma-2 protein . 22 ROS and the apoptotic cell death machinery . 27 iii 4.1 ROS as biphasic regulators of apoptosis . 28 4.2 Oncogene-mediated pro-oxidant state – Bcl-2 as a prime example 31 Protein Phosphatase 2A 32 5.1 The tumour suppressive role of PP2A 34 5.2 PP2A and the apoptotic cell death machinery . 36 Aims and Objectives . 38 MATERIALS AND METHODS 39 Cell lines and cell culture . 39 Reagents and chemicals 39 Antibodies . 40 Plasmids and SiRNAs . 41 Calcium phosphate-based transfection of adherent cells 42 Electroporation-based transfection of Jurkat cells . 43 Determination of protein concentration 44 Western blot analysis 44 Co-immunoprecipitation (co-IP) assay . 45 10 Mitochondrial-cytoplasmic fractionation . 46 11 Immunofluorescence confocal microscopy 47 12 MTT cell viability assay . 49 13 Crystal violet cell viability assay 49 14 Caspase activity assay . 50 15 Measurement of cellular O2- via Lucigenin chemiluminescence assay 51 16 Flow cytometry analysis of intracellular NO 51 17 Measurement of PP2A activity (serine/threonine phosphatase assay) . 52 18 Construction of Y289F-B56δ mutant via site-directed mutagenesis 54 iv 19 Extraction and purification of primary lymphoma tumours . 56 20 Statistical analysis . 57 RESULTS . 58 O2- promotes tumour chemoresistance by inducing Serine70 Bcl-2 phosphorylation . 58 1.1 Diethyldithiocarbamate (DDC) induced a parallel increase in both intracellular O2- and S70 pBcl-2 . 58 1.2 Scavenging of O2- abrogated DDC-induced S70 bcl-2 phosphorylation 61 1.3 SiRNA-mediated downregulation of SOD1 mimicked the effect of DDC . . 63 1.4 Treatment of bovine SOD1 led to a reduction in S70 pBcl-2 . 65 1.5 Pre-treatment of DDC promoted chemoresistance in tumour cells 67 1.6 Pre-treatment of DDC abated etoposide- and doxorubicin-induced activation of caspase-9 and caspase-3 69 1.7 Silencing of SOD1 protected against doxorubicin- and etoposide-induced cell death 71 1.8 S70 phosphorylation of Bcl-2 is required for the death-inhibitory activity of DDC . 72 1.9 S70 phosphorylation of Bcl-2 did not enhance its ability to sequester the pro-apoptotic protein, BAK . 75 O2- induced S70 pBcl-2 upregulation by inactivating the Bcl-2 phosphatase, PP2A 77 2.1 O2--induced S70 pBcl-2 upregulation is not a function of MAP kinases activation 77 2.2 O2- generated by pharmacological inhibition or genetic knockdown of SOD1 triggered a drop in PP2A activity 80 2.3 O2- inhibited the interaction between Bcl-2 and the catalytic subunit of PP2A . 82 2.4 Co-localization of PP2A-C and Bcl-2 was inhibited in cells with an augmented O2- level . 85 v 2.5 Mitochondrial translocation of PP2A-C was inhibited in tumour cells with heightened intracellular O2- level . 90 O2- augmented the level of S70 pBcl-2 by inhibiting the holoenzyme assembly of B56δ-containing PP2A (PP2AB56δ) . 94 3.1 The B56δ regulatory subunit of PP2A is responsible for the substrate recognition of Bcl-2 in Jurkat and MDA-231 cells 94 3.2 O2- inhibited the binding of the AC catalytic core, but not B56δ, to Bcl-2 . . 97 3.3 DDC treatment did not affect the co-localization of B56δ and Bcl-2 . 99 3.4 SiRNA-mediated knockdown of SOD1 inhibited the interaction of Bcl-2 with PP2A-C but not with B56δ 100 3.5 O2- inhibited mitochondrial localization of PP2A-AC catalytic core . 102 ONOO- as the missing link between O2- and the inhibition of PP2A 104 4.1 DDC treatment of tumour cells was accompanied by a drop in intracellular nitric oxide (NO) level . 104 4.2 ONOO- is necessary for DDC-induced S70 Bcl-2 phosphorylation . 106 4.3 Low doses of ONOO- mirrored the effects of O2- in promoting S70 pBcl-2 and chemoresistance . 107 4.4 ONOO- treatment mimicked the inhibitory effect of SOD1 downregulation on the interaction between B56δ and the AC catalytic core 110 4.5 O2- inhibited mitochondrial localization of PP2A-AC catalytic core via the production of ONOO- . 112 O2- inhibited PP2AB56δ holoenzyme assembly via ONOO--mediated nitration of B56δ at tyrosine-289 (Y289) . 113 5.1 Bioinformatics analysis revealed a conserved tyrosine residue on PP2AB56 regulatory subunits that is prone to nitrative modification . 113 5.2 DDC treatment augmented the level of 3-nitrotyrosine (3-NT) in HAB56α and HA-B56δ 117 5.3 3-NT level was elevated in endogenous B56δ upon DDC and ONOOtreatment . 119 vi 5.4 SOD1 knockdown induced B56δ tyrosine nitration and inhibited B56δmediated recruitment of PP2A-C to Bcl-2 . 121 5.5 Inhibition of NOX by diphenyliodium (DPI) abrogated the inhibitory effect of SOD1 knockdown on PP2AB56δ . 124 5.6 Scavenging of O2- via bSOD1 treatment potentiated PP2AB56δ mediated dephosphorylation of Bcl-2 at S70 . 126 5.7 DDC-induced O2- production also stimulated the phosphorylation of tau, but not pRb, and Raf 128 5.8 Nitration status of B56δY289 is positively correlated with the level of S70 pBcl-2 in Jurkat cells 130 5.9 Site-directed mutagenesis studies confirmed the regulatory role of Y289 B56δ nitration in O2--induced upregulation of S70 pBcl-2 133 5.10 Y289F-B56δ transfection restored the interaction between B56δ and PP2A-C in DDC-treated Jurkat cells 136 5.11 Y289F-B56δ transfection restored the co-localization status of Bcl-2 and PP2A-C in DDC-treated Jurkat cells 139 5.12 Y289F-B56δ transfection abolished the death-inhibitory effect of DDC in Jurkat T-leukemic cells 144 Clinical relevance of SOD1 downregulation in primary lymphoma biopsy samples . 146 6.1 An inverse relationship exists between SOD1 and S70 pBcl-2 in primary lymphoma cells 146 6.2 Primary lymphoma tumours expressing high level of S70 pBcl-2 harboured B56δ that are tyrosine nitrated 149 DISCUSSION . 151 ROS as intricate regulators of cell death 151 1.1 S70 pBcl-2 as a key player in O2--mediated chemoresistance 151 1.2 O2- and ONOO- – Killers or protectors? 152 1.3 SOD1 – are they tumour suppressors? 154 S70 pBcl-2 – How it is regulated, and how it regulates . 159 vii 2.1 Redox regulation of Bcl-2 phosphorylation 159 2.2 Potential source(s) of O2- and NO responsible for the induction of S70 pBcl-2 . 160 2.3 The death-regulatory role of S70 pBcl-2 165 A new facet in the anti-apoptotic activity of Bcl-2 170 Inhibitory tyrosine nitration of B56 regulatory isoforms – potential implication(s) beyond the activation of Bcl-2 . 172 4.1 Redox regulation of PP2AB56δ – implications on phospho-tau 172 4.2 B56 regulatory subunits as strategic targets for O2--mediated carcinogenesis 173 4.3 The fate of B56δ-dissociated PP2A-C 175 O2--mediated inhibition of PP2A – its relevance in a clinical setting 177 5.1 Is PP2A a druggable target for anti-cancer therapy? . 177 5.2 Activating specific PP2A complexes as a novel chemotherapeutic strategy . 180 5.3 Targeting intracellular O2- as a novel therapeutic strategy against Bcl-2induced chemoresistance 182 5.4 SOD1, S70 pBcl-2 and B56δY289 nitration (nitro-B56δY289) – a potential prognostic signature for better cancer management and care 183 Conclusion . 186 REFERENCES 189 APPENDICES 209 List of Publications 209 Conference Papers . 209 viii 91. 92. 93. 94. 95. 96. 97. 98. 99. 100. 101. 102. 103. 104. 105. 106. 107. 108. 109. 110. 111. 112. 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Pervaiz, Regulation of mitochondrial metabolism: yet another facet in the biology of the oncoprotein Bcl-2. Biochem J, 2011. 435(3): p. 545-51. Coombs GS, Schmitt AA, Canning CA, Alok A, Low IC, Banerjee N, Kaur S, Utomo V, Jones CM, Pervaiz S, Toone EJ, Virshup DM , Modulation of Wnt/betacatenin signaling and proliferation by a ferrous iron chelator with therapeutic efficacy in genetically engineered mouse models of cancer. Oncogene, 2012. 31(2): p. 213-25. Conference Papers Low, I.C. and S. Pervaiz, Bcl-2 modulates resveratrol-induced ROS production by regulating mitochondrial respiration in tumor cells. European Molecular Biology Organization (EMBO) workshop on Mitochondria, Apoptosis & Cancer (MAC), Prague, Czech Republic. * Low, I.C. and S. Pervaiz, Bcl-2 as a regulator of mitochondrial respiration in tumor cells. International Cell Death Society annual meeting - South African chapter, Cape town, South Africa. ** Low, I.C. and S. Pervaiz, Nitration-mediated inhibition of Protein Phosphatase 2A as the mediator of superoxide-induced Bcl-2 phosphorylation. 18th Annual General Meeting of the Society for Free Radical Biology and Medicine (SFRBM), Atlanta, USA. *** * Recipient of Best Poster Award. ** Presented a short talk. *** Received travel award and was selected for a talk at the opening session. 209 [...]... Piperazine-N,N′-bis (2- ethanesulfonic acid) PKB/Akt Protein kinase B PKC Protein kinase C PMSF Phenylmethylsulfonyl fluoride PP1 Protein phosphatase 1 PP1-C Catalytic subunit of PP1 PP2A Protein phosphatase 2A PP2A- A A scaffolding subunit of PP2A PP2AB56α B56 -containing PP2A enzyme PP2AB56γ1 B56 1-containing PP2A holoenzyme PP2AB56δ B56 -containing PP2A PP2A-C Catalytic subunit of PP2A pRb Retinoblastoma protein Prx... xanthine and it is unlikely to be a major source of cellular H2O2 In fact, a major portion of cellular H2O2 is contributed by the dismutation of O2- into H2O2 by the superoxide dismutase (SOD) enzymes A total of three isoforms have been identified in eukaryotic cells to date, all of which are extremely efficient in their catalytic conversion of O2- into H2O2 Thus, with the constant production of O2-... contributing to the pool of cytosolic O2- in the cells 1.1 .2. 3 Xanthine oxidase and other potential sources of intracellular O2- Xanthine oxidase is an enzyme involved in the catabolism of purine where it catalyses the oxidation of hypoxanthine to xanthine, and subsequently to uric acid [35] During this process, O2 is being reduced into both O2- and H2O2 [1, 35] However, studies have shown that the majority... phosphorylation of Bcl- 2 did not affect Bcl- 2- Bak interaction 76 Figure 10: DDC-induced upregulation of S70 pBcl -2 is not a function of MAP kinases activation 79 Figure 11: PP2A activity declined upon pharmacological inhibition or genetic silencing of SOD1 81 Figure 12: DDC inhibited the interaction between PP2A- C and Bcl- 2 84 Figure 13: DDC inhibited the co-localization of PP2A- C... arsenal of antioxidant machineries capable of eradicating H2O2 2. 2 Catalase Catalase is a haem-protein responsible for the neutralization of intracellular H2O2 [87, 88] It is widely expressed in most tissues, particularly in the liver Like SOD, catalase catalyses a dismutation reaction, whereby one H2O2 molecule is reduced to H2O while another H2O2 molecule is simultaneously oxidized to form O2 in a single... B56 and Bcl- 2 was not affected by DDC treatment 99 xii Figure 19: Silencing of SOD1 inhibited PP2A- C -B56 interaction, but not the interaction between Bcl- 2 and B56 101 Figure 20 : SiRNA -mediated knockdown of SOD1 inhibited mitochondrial translocation of PP2A- A and PP2A- C, but not B56 103 Figure 21 : DDC treatment resulted in a decline in intracellular NO level 105 Figure 22 : FeTPPS... O2- from the mitochondria as well as other enzymatic sources, it is of no surprise that the majority of cellular H2O2 stems from the dismutation of these O2- by the SODs Due to their role in the removal of O2-, SODs are considered as part of the cellular antioxidant machinery, and for this reason, a more detailed discussion of the enzyme in the following chapter on cellular antioxidant systems is warranted... it [1] 1 .2. 2 Sources of intracellular H2O2 Unlike the production of O2- by the NOX complexes, no enzyme is known to intentionally produce H2O2 in the cell In vivo, H2O2 are typically produced as byproducts of the various biological processes catalysed by the oxidase enzymes For instance, the mitochondrial monoamine oxidases are amongst the major contributors of mitochondrial H2O2 The monoamine oxidases... and Bcl- 2 87 Figure 14: SiRNA -mediated silencing of SOD1 inhibited the co-localization of PP2A- C and Bcl- 2 89 Figure 15: DDC treatment inhibited mitochondrial translocation of PP2A- C 93 Figure 16: HA -B56 interacts with Bcl- 2 96 Figure 17: DDC treatment inhibited the interaction of PP2A- C with the B56 regulatory subunit 98 Figure 18: Co-localization of B56 ... DDC-induced upregulation of S70 pBcl -2 106 Figure 23 : Low doses of ONOO- induced the phosphorylation of Bcl- 2 at S70 108 Figure 24 : Low doses of ONOO- protected against drug-induced cell death 109 Figure 25 : ONOO- treatment inhibited the interaction of Bcl- 2 with the AC catalytic core but not with the B56 regulatory subunit 111 Figure 26 : Pre-treatment of FeTPPS blocked the . subunit of PP1 PP2A Protein phosphatase 2A PP2A- A A scaffolding subunit of PP2A PP2A B56 B56 -containing PP2A enzyme PP2A B56 1 B56 1-containing PP2A holoenzyme PP2A B56 B56 -containing. level of S70 pBcl -2 by inhibiting the holoenzyme assembly of B56 -containing PP2A (PP2A B56 ) 94 3.1 The B56 regulatory subunit of PP2A is responsible for the substrate recognition of Bcl- 2 in. knockdown of SOD1 triggered a drop in PP2A activity 80 2. 3 O 2 - inhibited the interaction between Bcl- 2 and the catalytic subunit of PP2A 82 2. 4 Co-localization of PP2A- C and Bcl- 2 was inhibited